Process for preparing and cultivating hematopoietic progenitor cells

ABSTRACT

Process for the in vitro production of non-immortalised haematopoietic progenitor cells of the erythroid lineage, in which a population of erythroid progenitor cells is exposed to a combination of growth factors containing a glucocorticoid and an oestrogen and at least one ligand of a tyrosine kinase receptor at least until the cells begin to renew themselves.

FIELD OF THE INVENTION

The present invention relates to a) process for the preparation and invitro cultivation of haematopoietic progenitor cells, particularly ofthe erythroidal series.

DESCRIPTION OF RELATED ART

During normal haematopoiesis, pluripotent stem cells develop intoprogenitor cells which are intended for a specific developmental series(these progenitors are referred to as "committed"); these cells arethought to differ from the pluripotent stem cells in two respects:firstly, they are restricted in their ability to differentiate into asingle developmental series or a small number of specific developmentalseries. Secondly, these committed progenitor cells are generally thoughtto be either incapable of replicating continuously without simultaneousdifferentiation (this property is also referred to as the capacity forself renewal) or they do so only transiently (Till and McCulloch, 1980).It is therefore assumed that the progenitor cells, committed to aspecific developmental series, begin a predetermined programme ofchanges in gene expression which ends with the formation of a terminallydifferentiated cell. Pluripotent stem cells, on the other hand, arethought to retain their capacity to undergo numerous cell divisionswithout changing their state of differentiation or gene expression. Theprogramme which the progenitor cells undergo is obviously compatiblewith undergoing numerous cell divisions, but it is assumed that duringeach division the cells will undergo changes, however slight, in theirstate of differentiation or gene expression (Keller, 1992).

This view that a fixed determination/differentiation programmedetermines the development of the committed progenitor cells hasrecently been called into question in various ways: firstly, someobservations lead one to assume that normal committed progenitors canundergo extended phases of expansion, indicating self-renewal or relatedprocesses. Murine B-lymphocyte progenitors renew themselves constantlyunder a series of culture conditions (stromal feed cell layers plusinterleukin 7), but differentiate under other conditions into matureB-cells (Rolink et al., 1991). Similarly, individual murine granulocytemacrophage colony forming cells (GM-CFC), depending on the concentrationof GM-CSF, may produce between 100 and more than 10,000 maturegranulocytes and macrophages (Metcalf, 1980).

Another phenomenon which is difficult to reconcile with a fixedprogramme of the development of committed progenitors consists ofleukaemias. Although in some cases these start from pluripotent stemcells, other leukaemias clearly derive from committed progenitors(Sawyers et al., 1991). Regarding the latter type there is a frequentlyexpressed concept that the genetic changes which occur in leukaemiacells give them the abnormal ability of self-renewal, a quality whichthe corresponding normal progenitor cell does not have. Whereas in thechronic phase of chronic myeloid leukaemia (CML) clones of altered,multi-potent progenitor cells overgrow the corresponding normal clones(possibly on account of their greater capacity for self-renewal) othermutations which take place during the blast crisis lead to a massiveoutgrowth of immature progenitors and maturing cells of a specialdevelopment series, which is interpreted as self-renewal of abnormalcommitted progenitors (Daley et al., 1990; Elefanty et al., 1990;Kelliher et al., 1990).

Recently, it was shown, in chicken cells, that normal haematopoieticprogenitors which are committed to the erythroid developmental seriesare capable under certain conditions of sustained self-renewal(Schroeder et al., 1993; Hayman et al., 1993). It was shown that thecombined effect of TGFα (Transforming Growth Factor, a ligand for thechicken homologue of epidermal growth factorreceptorlc-erbB-protooncogene (TGFαR/c-erbB; Lax et al., 1988) andoestradiol induced the outgrowth of normal progenitors from chicken bonemarrow. These cells are known as SCF/TGFα-progenitor cells on account oftheir ability to grow out from cultures which contain TGFα plusoestradiol or SCF (stem cell factor) (cells which grow in the presenceof SCF are termed SCF progenitor cells). SCF/TGFα-progenitor cellsexpress the c-kit-protooncogene, the oestradiol receptor andTGFαR/c-erbB and are capable of sustained self-renewal in the presenceof TGFα plus oestradiol until the end of their normal in vitro life. Ithas also been shown that erythroid progenitors which cannot bedistinguished from normal CFU-Es (colony-forming unit erythroids) interms of all the properties investigated (known as SCF-progenitors)could be cultivated from bone marrow using chicken-SCF (Hayman et al.,1993). By contrast to the SCF/TGFα progenitors with the capacity forself-renewal, the SCF progenitors lacked the expression of TGFαR/c-erbB,and in the presence of SCF the cells exhibited only transientself-renewal during the period of 7 to 10 days. When they were switchedto differentiation factors (erythropoietin plus insulin), both typesdifferentiated with indistinguishable kinetics in erythrocytes. Thisindicated that the SCF/TGFα-progenitors are not the progenitors of SCFprogenitors as was originally assumed on account of the fact thatSCF/TGFα-progenitors are relatively rare (1 in 15,000 normal bone marrowcells), whereas the SCF-progenitors are much more common (1 in 300-500;Hayman et al., 1993). However, these results failed to answer thequestion as to whether the self-renewing SCF/TGFα-progenitors derivedfrom even more immature progenitors. One possible answer is that thesecells constitute a separate, rare cell type which occurs in the bonemarrow and develops from multipotent progenitors like a separate cellline. One alternative answer would be that these cells derive fromnormal CFU-Es which acquire the potential for self-renewal only underthe effect of specific combinations of growth factors and hormones whichare not normally active in erythropoiesis.

In earlier studies (Schroeder et al., 1993) it was shown that there aretwo fundamental requirements for the outgrowth of SCF/TGFα-progenitorsfrom bone marrow: firstly, a specific length of time--the outgrowthnever occurred until 11 to 14 days had passed; secondly, the dependencyon both TGFα and oestradiol, which was demonstrated by the fact that theoutgrowth of the cells was completely inhibited by an oestradiolantagonist and did not occur in the absence of TGFα. If the first answeris correct and SCF/TGFα-progenitors are a particular cell type which isalways present in normal bone marrow and is dependent only on TGFα andoestradiol, other factors should not have any significant effect on thefrequency of these cells; however, two observations would tend to speakagainst this simplified model: firstly, it has been found that theoutgrowth of SCF/TGFα-progenitors was strongly inhibited in the presenceof chicken serum which had been treated with animal charcoal, whereas inFreon-treated or untreated serum it was not substantially affected. Thisleads one to assume that in addition to TGFα and oestradiol, otherfactors which are eliminated by the animal charcoal treatment have aneffect on the SCF/TGFα-progenitors at some stage of their formation. Ithas also been observed that bone marrow cells kept in SCF plusoestradiol were stationary after 8 to 10 days but started to grow slowlyagain on approximately the 14th day. These cells expressed TGFαR/c-erbBin a relatively high concentration (Hayman et al., 1993) and could begrown in TGFα plus oestradiol, which leads one to suppose that thesecells are SCF/TGFα-progenitors which had grown out of the originalpopulation of SCF-progenitors.

SUMMARY OF THE INVENTION

The aim of the present invention is to clarify the mechanisms which areinvolved in the formation of haematopoietic progenitors of the erythroidlineage, which express c-Kit and TGFαR/c-ErbB (referred to within thescope of the present invention as "SCF/TGFα-progenitors"), and on thebasis of the knowledge obtained, to prepare a process for cultivatingnormal erythroid progenitor cells in vitro.

In particular, the intention is to provide a process which makes itpossible to mass produce non-immortalised and hence geneticallyunaltered human haematopoietic progenitor cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Within the scope of the present invention it was first shown, regardingchicken cells, that in the presence of SCF, TGFα, oestradiol andspecific unidentified chicken serum factors SCF/TGFα-progenitors developin cultures of purified SCF-progenitors.

It has been shown that when SCF-progenitors are cultivated in thepresence of a combination of SCF, TGFα, oestradiol and initiallyundefined factors from normal or anaemic chicken serum, a largeproportion of these cells undergo neither differentiation nor apoptosis,but begin to express increasing quantities of TGFαR/c-erbB in a strictlytime-dependent pattern, resulting in the production ofSCF/TGFα-progenitors after 10 to 14 days. At this time, the expressionof TGFαR/c-erbB in the cells is obviously high enough to allowproliferation-in the presence of TGFα and oestradiol in the absence ofSCF. When specially treated chicken sera were used it was shown that noSCF/TGFα-progenitors are formed if one of these three factors (SCF, TGFαor oestradiol) was missing. On the other hand, the formation of theproliferating progenitors in the presence of SCF, TGFα and oestradiolwas partly inhibited, although not eliminated, if the unidentifiedactivity of the chicken serum was absent. Further tests on a chickenmodel showed that the initially unidentified activity can be at leastpartly replaced by two defined factors: 1. theglucocorticoid-receptor-ligand dexamethasone and 2. the tyrosinekinasereceptor-ligand insulin-like growth factor 1 (IGF-1). It could also beassumed that erythropoietin is another factor which is responsible forthe activity in the chicken serum.

When SCF-progenitors were cultivated in SCF, TGFα and oestradiol,SCF/TGFα-progenitors became concentrated in the culture, until afterabout two to two and a half weeks they were predominant in the culture.The expression of TGFαR/c-erbB increased with time when theSCF-progenitors were cultivated in SCF, TGFα and oestradiol. On thebasis of the mass culture experiments carried out, it was initiallyimpossible to distinguish between two possible ways in which theSCF/TGFα-progenitors had formed from the cultures of SCF-progenitors.The first (trivial) possibility would be that a small number ofSCF/TGFα-progenitors which express c-erbB exist in normal bone marrowand hence in SCF-progenitor populations from the outset, these cellsgradually overgrowing the SCF-progenitors (if the culture is carried outin the presence of SCF, TGFα and oestradiol, which might possibly helpto give these cells a growth advantage). The more interesting, untrivialpossibility, however, is that the bone marrow does not contain, at theoutset, any erythroid progenitors capable of proliferating in TGFα andoestradiol alone, but that SCF/TGFα-progenitors are induced to developfrom SCF-progenitors if all three factors plus certain chicken serumcomponents or dexamethasone and IGF-1 (see above) are present.

The experiments carried out within the scope of the present inventionshowed that this latter hypothesis is correct, i.e. thatSCF/TGFα-progenitors can develop from SCF-progenitors. The resultsobtained with chicken cells showed that normal erthyroid progenitors(SCF-progenitors which resemble the colony forming unit erythroid(CFU-E) progenitor in all the qualities investigated) develop under thecontrol-of at least two growth factors (SCF, TGFα) plus a steroidhormone (oestradiol) and an initially undefined activity from chickenserum, which was later partially identified as dexamethasone plus IGF-1,into another type of erythroid progenitor (SCF/TGFα-progenitor). Thisother type of progenitor is characterised by its newly acquiredexpression of TGFαR/c-erbB (which corresponds to the mammalian EGF/TGFαreceptor) and its ability to undergo sustained self-renewal as areaction to TGFα and oestradiol. The differentiation programme of theSCF/TGFα-progenitors after treatment with differentiation factors (EPO,insulin) strongly resembles the normal CFU-E-progenitor (Hayman et al.,1993).

Since it has hitherto been assumed that erythroid progenitors areirreversibly committed to differentiation and undergo a fixed programmeof 5 to 10 cell divisions, the finding obtained with chicken cells thatthey may acquire a self-renewal potential under certain conditions bychanging their differentiation programme ("developmental switch")was ofconsiderable interest, provided that these findings were valid formammalian or even human cells. Within the scope of the present inventionit is shown that the results of the chicken system are applicable tohuman cells to a surprising degree.

There is a need for human haematopoietic progenitor cells which can becultivated in vitro particularly for the purposes of transplanting suchcells in the treatment of cancer and AIDS patients. A further use ofsuch transplantation is the treatment, by gene therapy, of chronicanaemias in which the maturing of the erythrocytes is disrupted, e.g.thalassaemia and other genetically caused anaemias.

One of the few definite preconditions required for successfultransplantation of blood cells is the expression of CD34. However, it isnot known at which stage of development the subpopulation of the CD34⁺-cells is at, which is actually responsible for successfultransplantation, although it is assumed that the developmental seriesand stage of differentiation of the cells play a part.

Autologous or allogenic transplantation of haematopoietic progenitorcells involves difficulties, one of the main problems being that asufficient number of cells having the proliferation potential necessaryfor successful reconstitution of the haematopoietic system has to betransplanted and the criteria which determine this potential have notbeen adequately researched as yet.

Hitherto, for allogenic transplantations, bone marrow cells from healthydonors are frequently used; for autologous transplants, stem cells fromperipheral blood are used which are mobilised during the recovery of thepatient from chemotherapy and/or by treatment with recombinant growthfactors. These methods are expensive; in addition, they involveconsiderable unpleasantness for the donor or yield poor results owing tohaematological changes in the patient. It has therefore recently beenproposed as an alternative that stem cells be used from cytokine-treatedhealthy donors.

Another alternative regarded as highly promising is to use umbilicalcord blood cells instead of bone marrow or CD34⁺ -positive cells fromperipheral blood, as the majority of haematopoietic stem and progenitorcells from umbilical cord blood are at an earlier stage of developmentand have a greater proliferation potential. However, since about 1.5litres of umbilical cord blood would be necessary for transplanting inan adult, the requirement being 5×10⁵ -2×10⁶ CD34⁺ -cells per kg of bodyweight, this method is subject to limits in the treatment of adults.

There is therefore a need for a process which permits mass culture ofautologously or allogenically transplantable haematopoietic progenitorcells.

Within the scope of the present invention it has been shown that humanerythroid progenitor cells, surprisingly, exhibit similar behaviour tothe corresponding chicken cells, in that they undergo a change in theirdifferentiation programme as a result of which they acquire a potentialfor self-renewal. Like the chicken cells, the cells required SCF,oestradiol and dexamethasone in order to acquire the ability forsustained self-renewal. IGF-1 had a positive influence on the growth ofchicken and human erythroblasts. Some of the cells in the culture ofhuman erythroid progenitors, which was obtained within the scope of theexperiments carried out, reacted to ligands of the EGF-receptor, whichis an additional indication that the human cells are similar in theirbehaviour to the chicken SCF/TGFα-progenitors under the influence ofcertain growth and differentiation factors.

The present invention is thus also based on the critical realisationthat a change in the differentiation programme of human erythroidprecursors must take place, on the basis of which change they acquirethe ability for sustained outgrowth. This change in the differentiationprogramme should be induced by the interaction of factors which areligands of representatives of the same receptor groups the activation ofwhich induces the development of self-renewing erythroid progenitorsfrom chicken bone marrow.

The invention thus relates to a process for in vitro production ofnon-immortalised haematopoietic progenitor cells of the erythroidlineage, which is characterised in that cells containing a population oferythroid progenitors, in a medium which contains the usual componentsnecessary for the growth of erythroid cells, are exposed to acombination of growth factors, containing at least one ligand of theoestrogen receptor and at least one ligand of the glucocorticoidreceptor and at least one, preferably at least two ligands of atyrosine-kinase receptor, at least until the cells begin to renewthemselves, and subsequently, if desired, the cells are further culturedin a medium which contains the factors required for sustainedself-renewal.

By treating the cells with the combination of growth factors(hereinafter referred to as "factor combination") the cells undergo achange in the differentiation programme. This is accompanied by a changein the expression pattern of the receptors which are newly expressed orhighly regulated by the action of the factor combination, and/or bychanging the expression pattern of protein components of the cell signaltransmission pathways triggered by these epigenetic changes.

The term "self renewal" refers to the ability of cells to form daughtercells which do not mature measurably during the subsequent celldivisions, i.e. in which there is no measurable further accumulation ofthose proteins which are typical of the mature cells but may also beexpressed in small amounts in progenitor cells. Another importantcriterion for self-renewal is that the ratio of proteins of the mature(terminally differentiated) cell (e.g. haemoglobin) and proteins whichare necessary for the function of each cell (so-called "housekeepingproteins", e.g. glycolytic enzymes) does not measurably change.

Preferably, the process according to the invention is applied to humancells.

The starting cell material used is preferably a cell population of bonemarrow, peripheral blood or, in a particularly preferred embodiment,umbilical cord blood containing a concentration of CD34-positive cells.Concentration may be achieved by methods known from the literature; asurvey of such methods is given in the textbook "Hematopoietic StemCells, The Mulhouse Manual", 1994.

The cells are cultivated in vitro at least until self-renewal occurs.Purely externally, cells with potential for self-renewal can berecognised by the fact that they are continuously dividing in theculture, i.e. proliferating exponentially, for a period of timecorresponding to the in vitro life of the cells (50-70 generations inthe case of human cells) or part of this life, and they have a constantsize and a comparatively small content of erythrocyte proteins (e.g.haemoglobin). Anyone skilled in the art can tell from these criteria, inpreliminary tests, the point in time in which the cells acquired apotential for self-renewal and can accordingly define the duration ofcultivation.

The self-renewal of the human cells of the erythroid lineage which areobtainable within the scope of the present invention is characterised inthat the cells divide without any appreciable differentiation over asubstantially longer period of time than has hitherto been shown fornormal human BFU-Es (burst forming unit erythroids).

The factor combination is preferably a combination of at least three andpreferably at least four factors, at least two of them being ligands oftyrosine kinase receptors. There is a quantity of literature onreceptors of this type, the families and sub-families to which theybelong, their ligands and the signal transmission pathways triggered bytheir activation and new examples are constantly being identified. Whatis common to the tyrosine kinase receptors is the fact that after thebinding of their ligand they themselves phosphorylate to tyrosines.After this autophosphorylation the phosphotyrosine groups interact withspecific cytoplasmatic molecules, thereby triggering the cell responseto the growth factors.

The family of the tyrosine kinase receptors is divided into variousclasses and sub-families; these include the class to which the EGFRfamily, HER2/neu/c-erbB-2 and HER3/c-erbB-3 belong; the class to whichthe insulin receptor, the insulin related receptor and the IGF-1receptor belong; the class which comprises PDGF receptor, PDGFβreceptor, MCSF-1 receptor and c-kit; the class of the fibroblast growthfactor receptors (FGF-receptor1, FGF receptor2, FGF receptor3, FGFreceptor4) and the HGFR receptor (hepatocyte growth factor receptor).Some of these classes share the feature that the kinase domain isinterrupted by a sequence. Regarding the tyrosine kinase receptors andtheir ligands, we refer to the summarising article by Fantl et al., 1993and Van der Geer, 1994, including the literature specifically citedtherein regarding the individual receptors.

The factor combination of tyrosine kinase receptor ligands consists ofat least one ligand for receptors from various families within thetyrosine kinase receptors. An example of such a combination is

i) at least one ligand of a tyrosine kinase receptor which has acontinuous kinase domain, and

ii) at least one ligand of a tyrosine kinase receptor which has a kinasedomain interrupted by an insert.

Examples of representatives of the receptors defined in i) are themembers of the EGF-receptor family (Human Epidermal Growth FactorReceptor 1-4); other only partly identified receptors belong to thisfamily.

Ligands of the receptors defined in i) include, inter alia, EGF, TGFα,NDF (Neuronal Differentiation Factor; Peles and Yarden, 1993), includingthe variants produced by differential splicing, Heregulin, Amphiregulin,Glial Growth Factor etc. (Fantl et al., 1993).

Ligands of the receptors defined in ii) include, inter alia, the c-kitligand SCF (Stem Cell Factor), Platelet Derived Growth Factor (PDGF)alpha and beta, all the members of the fibroblast growth factor family,CSF-1 (Colony Stimulating Factor 1) and vascularising factors (e.g.VEGF, Vascular Endothelial Growth Factor) (Fantl et al. 1993).

In addition, there are a plurality of tyrosine kinase receptors whichcannot be clearly allocated to these two groups (the ligands of whichare only partly known), activation of which by the corresponding ligandsmay cause the outgrowth of human progenitor cells; the correspondingligands may also be used within the scope of the present invention.These receptors include: Hepatocyte Growth Factor Receptor (the ligandof which is also known as "Scatter factor"; the findings obtained byGalimi et al., 1994 indicate that the Hepatocyte Growth Factor Receptor(HGFR), which is assumed to activate the same signal transmissionpathways as the EGF receptor, plays an important part in CD34⁺ cells andhuman erythroid progenitor cells produced therefrom), c-sea and c-ros(the ligands of which have not yet been identified), various epithelialcell-specific receptors the ligands of which are unknown, a group ofreceptors cloned from erythroid cells which have recently been described(e.g. by Tamagnone et al., 1993 and Kaipainen et al., 1993), the ligandsof which are also as yet unknown, and also the members of theneurothrophin receptors (trk, trk-B, trk-C with the ligands NGF, BNDF,etc.), and receptors of the insulin receptor family (insulin receptor,IGF-1 receptor etc.).

Without wishing to be tied to the theory, it would appear to beessential, for triggering the change in the differentiation programme,that different signal transmission pathways are set in motion by thebinding of the ligands and the consequent activation of the receptorsdefined in i) and ii).

Apart from the two ligands of the tyrosine kinase receptors, the factorcombination contains:

iii) at least one ligand of the oestrogen receptor and at least oneligand of the glycocorticoid receptor.

Within the scope of the present invention, natural or syntheticallyproduced steroid hormones which, like oestradiol, activate the oestrogenreceptor or, like hydrocortisone, activate the glycocorticoid receptor,are suitable.

Additionally, the factor combination may possibly contain ligands of theprogesterone receptor, such as aldosterol and progesterone.

What is common to these hormones is that a) they are low molecular, b)they bind to receptors located in the nucleus which constitutetranscription factors regulated in their activity by the hormone(proteins which alter the genes in their activity) and c) they arecapable of changing the differentiation programme of cells in some ofthe systems investigated hitherto.

Within the scope of the present invention, it has been found that, apartfrom an oestrogen, dexamethasone, a glucocorticoid, in particular is ofcrucial importance in the outgrowth of self-renewing chicken and humanerythroid progenitor cells.

In addition, the factor combination iv) may contain one or moreadditional factors.

The additional factors iv) may be in particular ligands which at leastspeed up the change in the differentiation programme and hence bringabout a more efficient outgrowth of the cells. These factors aregenerally added to the medium right at the beginning of the culture,whilst it should be borne in mind that different factors may benecessary at different times during the change to the differentiationprogramme. With regard to accelerating the change in the differentiationprogramme, therefore, it may be advisable to eliminate from the mediumany factors which are essential for triggering this process but arelater inessential or even disadvantageous, at a suitable point in timewhich may be determined by a series of tests. Additional factors whichmay be considered are as follows:

1. Ligands of receptors which act by serine phosphorylation of targetproteins. (TGFβ-receptor family). In particular, the ligands activin,inhibin, BMP etc. which play a part in early embryonic development areimportant (Laufer, 1993; Hogan, 1993).

2. Ligands of other tyrosine kinase receptors, particularly IGF-1 orhepatocyte growth factor (HGF).

3. Representatives of the large group of cytokines or interleukins(growth and differentiation factors in the haematopoietic and immunesystem). Virtually all these cytokines bind to receptors which do notthemselves have any known enzyme activity, but some of the receptorsform complexes with intracellular tyrosine kinases. A summary of thisconstantly growing family of receptors and their ligands is provided inBoulay and Paul, 1993.

An essential feature for the activity of a cytokine which can be usedwithin the scope of the present invention is that first of all itstimulates the proliferation of immature progenitors and secondly doesnot have any effect which negatively influences cell growth and/ortriggers apoptosis (programmed cell death). Within the scope of thepresent invention, preferred cytokines are IL-1, IL-3, IL-11 and IL-13.EPO is particularly preferred.

The population of cells obtained by the action of the combination offactors may be frozen after the start of self-renewal and thawed asnecessary and thereafter either further cultivated or transplanteddirectly in order to make use of the self-renewal potential of thecells, acquired in vitro, for proliferation in vivo.

However, the cells may be cultivated beyond the length of time duringwhich they acquire the self-renewal potential, so as to obtain a largernumber of proliferating cells within the population.

Further cultivation of the proliferating cells is carried out in thepresence of those growth and differentiation factors which the cellsrequire for sustained self-renewal.

For chicken cells, TGFα is one of the factors needed for the sustainedself-renewal potential and hence for the cultivation of the cells over afairly long period of time in order to obtain a large number of cells.For human cells, the factors preferably used for further cultivation ofthe cells are ligands of the type defined in i) such as EGF and/or TGFα,and/or HGF, as well as SCF, and also EPO and IGF-1.

The suitable combination of factors both for the induction of theself-renewal and for further cultivation of the proliferating cells isdetermined by testing the response of the cells and their growthcharacteristics under the effect of various mixtures of factors atvarious times; examples of such tests are described, inter alia, inExamples 4b), 5, 7b and 8. The factor mixture is preferably optimised byfirst testing various multi-component mixtures so as to identify themost effective mixture. Then, one factor is eliminated step by step fromthe most effective mixture and the behaviour of the culture with andwithout the factor is compared. To summarise, the factor combination isadjusted so as to achieve the most rapid and efficient outgrowthpossible for self-renewal of viable cells with the fewest possiblefactors.

The treatment with the combination of SCF, TGFα, oestradiol and anotheractivity, carried out within the scope of the present invention,resulted in an increase in the expression of biologically activeTGFαR/c-ErbB in chicken and human cells, which manifested itself inchicken cells by an increase in autophosphorylated receptor after theaddition of ligand; the further activity, in the case of chicken cells,was an unidentified activity in the chicken serum and, in the case ofhuman cells, EPO.

In one embodiment of the invention, the factor combination for thepreparation of human haematopoietic progenitor cells consists of

i) a ligand of a receptor from the family of the EGF receptors and/orthe HGF receptor,

ii) a ligand of c-Kit;

iii) oestradiol and dexamethasone, and

iv) erythropoietin and IGF-1.

In one particular embodiment of the invention

i) is EGF and/or TGFα and/or HGF, and

ii) is SCF.

If the factors are components of the medium in a sufficientconcentration, e.g. as serum components, they must not be addedseparately.

The usual components contained in the medium apart from the factorcombination and necessary for the growth of the cells, such as vitamins,amino acids, etc., are well known to those skilled in the art; they arecontained in commercially available media and may be found in therelevant textbooks, such as "Haematopoietic Stem Cells, The MulhouseManual", 1994 and specialist articles such as Sawada et al., 1990.

The cells obtained by the process according to the invention may, afterremoval of the culture medium, be suspended in a medium suitable fortherapeutic use, e.g. human serum albumin (HSA) or autologous plasma andused for allogenic or autologous transplantation. The process accordingto the invention may be used, inter alia, to cultivate haematopoieticcells from a supply of blood cells of an individual whose production ofCD34 positive cells has been stimulated, e.g. by treatment withcytokines, in the event of the need for a transplant. Thesehaematopoietic cells can be stored frozen, thawed when needed, amplifiedby culturing in vitro and used, optionally after suitable gene transfer,for therapeutic purposes in the patient.

An example of a strategy in which genetically altered human erythroidcells cultivated in vitro may be used is the treatment of sickle cellanaemia by gene therapy. This inherited disease occurs particularly inthe USA in a large number of coloured patients. One possible procedureconsists in cultivating erythroid progenitors from bone marrow,peripheral blood or (in the event of prenatal diagnosis) umbilical cordblood, gene transfer of the globin-gene locus which carries thehereditary persistence of foetal haemoglobin (HPFH) mutation andadministration of these genetically altered somatic cells (the germ pathis unaffected) to the patient.

Gene transfer into the cells obtained according to the invention may becarried out by standard methods for the transfection of such cells.These methods include gene transfer using viral vector (retrovirus,adenovirus, adeno-associated virus) or using non-viral systems based onreceptor-mediated endocytosis; summaries of conventional methods areprovided for example by Mitani and Caskey, 1993; Jolly, 1994; Vile andRussel, 1994; Tepper and Mule, 1994; Zatloukal et al., 1993, WO93/07283.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: outgrowth of SCF/TGFα-progenitors from SCF progenitors

FIGS. 2A, 2A-1, 2B, 2B-1, 2C, 2C-1, 2D, and 2D-1: Expression andbioactivity of c-Kit and c-ErbB during the proliferation ofSCF-progenitors in SCF, TGFα and oestradiol

FIGS. 3A-3C Change in growth factor dependency of SCF progenitors whichproliferate in SCF, TGFα and oestradiol

FIGS. 4A, 4A-1, 4B, and 4B-1: Experimental strategy for clarifying theformation of SCF/TGFα progenitors

FIGS. 5A and 5B: Development of SCF/TGFα progenitors from SCFprogenitors

FIGS. 6A-6C: LD clones grown in SCF, TGFα and oestradiol correspond toSCF/TGFα progenitors: expression of bioactive c-ErbB and proliferationreaction to TGFα

FIGS. 7A-7C: A factor in chicken serum facilitates the development ofSCF progenitors to SCF/TGFα progenitors

FIGS. 8A and 8B: Need for oestradiol and SCF during the development ofSCF/TGFα progenitors-from SCF. progenitors

FIG. 8C: Acceleration of the development of SCF to SCF/TGFα progenitorsusing anaemic chicken serum

FIG. 9A: Speeding up the conversion of SCF progenitors into SCF/TGFαprogenitors using dexamethasone

FIG. 9B: Definition of insulin-like growth factor (IGF-1) as one of thefactors responsible for the activity in chicken serum

FIGS. 10A and 10B: Outgrowth of erythroid cells from human CD34⁺ cellsfrom peripheral blood

FIGS. 11A-1, 11A-2, 11B, 11C, and 11D: Characterisation of humanerythroid progenitors cultivated in vitro

FIG. 12A: Prolonging the self-renewing potential of human CD34⁺ cellsfrom umbilical cord blood by means of dexamethasone

FIG. 12B: Increasing the growth of human cells from peripheral bloodusing IGF-1

FIG. 13: Comparing the properties of outgrowing human erythroidprogenitors with chicken cells

In the Examples which follow, the materials and methods described byHayman et al., 1993 were used unless otherwise stated. In Examples 1-6,chicken cells were used and in Examples 7 and 8 human cells were used.

EXAMPLE 1

Concentration of SCF/TGFα progenitors by cultivating SCF progenitors inSCF, TGFα and oestradiol

In order to determine systematically whether SCF progenitors containSCF/TGFα progenitors or whether they contain cells which can developinto this type of cell, cells from a 6 day old culture of purified SCFprogenitors (Hayman et al., 1993) were cultivated in CFU-E mediumcontaining 100 ng/ml of recombinant SCF, 5 ng/ml of TGFα and 5×10⁷ Moestradiol and the cell proliferation was monitored by counting usingthe system described by Hayman et al., 1993 (CASY-1, sharpness system).(The rationale behind using all three factors was to keep the SCFprogenitors alive as long as possible and at the same time stimulate thegrowth of any SCF/TGFα progenitors present or generated in the culture.)The results of this experiment are shown in FIG. 1. Surprisingly (and insharp contrast to the results obtained in a comparative test by growingthe same cells in TGFα plus oestradiol) the cells showed only a weak,transient decrease in their growth rate around day 8 to 10 but continuedto proliferate exponentially thereafter, with a doubling time of 18 to22 hours from day 25 to 30, after which they underwent senescence (FIG.1, open circles).

In order to determine whether the SCF progenitors which had beencultivated in SCF, TGFα and oestradiol contained SCF/TGFα progenitors orhad developed themselves, and to obtain a rough estimate of theirnumbers, aliquots of the culture were taken at different times, washed,transferred into CFU-E medium containing TGFα and oestradiol but no SCF,and the number of cells was compared with that of the culture whichcontained all three factors. (The cells were kept at a density ofbetween 1×10⁶ and 2×10⁶ per ml by suitable dilution with fresh mediumand the cumulative cell numbers were calculated from the numbers ofcells obtained and the corresponding dilution factors (Schroeder et al.,1993;

Hayman et al., 1993)) (FIG. 1, arrows). When the three-factorcombination was removed from the cells on day 5, they immediatelystopped proliferating. The numbers of cells remained approximatelyconstant up till day 11.

During this time, most of the cells underwent apoptosis (these were notdistinguished from the living cells by the cell counter), whilst somehealthy clumps remained, which began to form the culture around day 13to 14. After that, the cells underwent growth, in the presence of TGFαplus oestradiol, which was indistinguishable in its kinetics from thatof the control culture (FIG. 1, solid circles).

Different behaviour of the cells was observed when cells which had beencultivated in SCF, TGFα and oestradiol were transferred on day 12 intomedium containing TGFα plus oestradiol (FIG. 1, solid rectangles). Uptill this time, only some of the cells had suffered apoptosis whilstmany others carried on growing, which became apparent as a temporaryreduction in the growth rate between days 13 and 16. After that, thecells grew at similar speed in TGFα plus oestradiol to the controlcells. After 18 days culture, transfer of all three factors to TGFα plusoestradiol had no noticeable effect on the cell proliferation (FIG. 1,solid triangles), indicating that, at this time, the culture consistedentirely of SCF/TGFα progenitors.

EXAMPLE 2

Increase in the expression of bioactive TGFαR/c-erbB in SCF progenitorscultivated in SCF, TGFα and oestradiol

a) Expression and bioactivity of c-Kit and c-ErbB during theproliferation of SCF progenitors in SCF, TGFα and oestradiol

On the basis of the results obtained in Example 1, it was assumed thatthe SCF progenitors cultivated in SCF, TGFα and oestradiol were eitherovergrown by SCF/TGFα progenitors which were there from the outset orhad developed into such. The purpose of the experiments was to show thatthe proliferating cells actually express bioactive TGFαR/c-ErbB, whichshould become apparent in the biochemical reaction (autophosphorylation)or biological reactions (stimulation of proliferation in correspondingassays) to be expected. For this purpose, on days 6, 12 and 20, aliquotsof the culture and control culture (see Example 1) were taken, washed,incubated overnight in medium without growth factors, stimulated for 5minutes with various factor combinations and further treated asdescribed by Hayman et al., 1993 and used for the phosphotyrosine blotand the subsequent Western blot (using anti-TGFαR- orc-erbB-antibodies).

After a total of 6 days (after an initial 3 days in SCF) the cellscultivated in SCF, TGFα and oestradiol showed the clear phosphorylationof c-Kit expected on account of the reaction to SCF and they expressedlarge quantities of c-Kit. By contrast, according to Western blotanalysis, the cells contained only very tiny amounts of TGFαR/c-ErbB andthere was no visible autophosphorylation of c-ErbB (these experimentsare shown in FIG. 2A, the arrows indicating the 170 kd TGFαR/c-ErbBprotein whilst the tips of the arrows indicate the 140 kd SCF-R/c-Kitprotein; the black circle in the lower boxes indicates the position of abackground band without reference to TGFαR/c-ErbB).

After 11 days, the expression of TGFαR/c-ErbB had increasedsignificantly, as demonstrated by c-ErbB-Western blot. In addition, aweak, although significantly detectable reaction of autophosphorylationof the c-ErbB protein could be made out on the ligands (FIG. 2B-1). Asexpected, the cells were still expressing autophosphorylated c-Kit (FIG.2B-1).

Similarly, the cells tested after 20 days (at this time they weregrowing both in TGFα plus oestradiol and also in SCF, TGFα andoestradiol) expressed increased amounts of TGFαR/c-ErbB, which was nowclearly autophosphorylatable as a reaction to TGFα (FIG. 2C-1).Surprisingly, the cells were still expressing smaller amounts ofTGFαR/c-ErbB than the control cells which had been cultivated fromuntreated bone marrow in TGFα plus oestradiol. (Schroeder et al., 1993;Hayman et al, 1993). The results obtained show that SCF precursorscultivated in TGFα, SCF and oestradiol express small amounts ofTGFαR/c-ErbB even after 6 days and thereafter this expression increasescontinuously throughout the next 8 to 14 days.

b) Change in the growth factor dependency of SCF progenitors whichproliferate in SCF, TGFα and oestradiol.

In order to confirm that the biochemically detected TGFαR/c-ErbBactually constitutes the bioactive receptor, the cells were additionallytested by ³ H!-thymidine incorporation assay to their reaction to SCF,TGFα and oestradiol as described by Hayman et al., 1993.

In order to do this, aliquots of a culture of SCF progenitors (5 days,FIG. 3A) or of cells which had been cultivated for 11 or 20 days in SCF,TGFα and oestradiol (FIGS. 3B and 3C) were investigated for theirreaction to various factors (100 relative units correspond to the factorconcentrations given under the symbols). It is apparent that the cellscultivated for 6 days in SCF, TGFα and oestradiol reacted to SCF andoestradiol as expected, whereas there was no detectable reaction to TGFα(FIG. 3A). After 11 days in contact with these factors, the reaction ofthe cells to SCF and oestradiol was unchanged, but now a weak butdistinct reaction to TGFα could be made out (FIG. 3B). As expected, thecells cultivated for 20 days in SCF, TGFα an d oestradiol reactedstrongly to all three factors showing no difference from thecontrol-SCF/TGFα progenitors (FIG. 3C).

To summarise, the results obtained show that self-renewing SCF/TGFαprogenitors can be cultivated efficiently from erythroid progenitorswhich initially react only to SCF and which lack both detectablequantities of TGFαR/c-ErbB and also the ability for longer lastingself-renewal.

EXAMPLE 3

Development of SCF/TGFα progenitors from SCF progenitors

In order to clarify the question of the origin of SCF/TGFα progenitorsfrom cultures of SCF progenitors, the method of cloning by limitingdilution, hereinafter referred to as "LD cloning", was used. This methodmake s it possible to analyse the proliferation characteristics (anddifferentiation characteristics) of individual proliferating cells in acomplex mixture of non-proliferating cells, because at a suitabledilution it is possible to monitor the development of individualproliferating cells in individual wells of cell culture plates (96 wellplates). The success of such a method naturally depends on good cloningefficiency (10 to 50%) of the proliferating cells which are to beanalysed, a criterion which is satisfied if the number of proliferatingclones obtained is a linear function of the number of cells seeded out,up to a very few (1 to 10) clones per 96 well plate. (The fact that thiscriterion is satisfied for clarification of the present question hasbeen demonstrated for SCF and SCF/TGFα progenitors by Hayman et al.,1993.)

The question as to how the LD cloning occurs between the two possiblemodels (selective outgrowth of rare SCF/TGFα progenitors from SCFprogenitors or development of SCF progenitors into SCF/TGFα progenitors)is illustrated in FIGS. 4A-1 and 4B-1. FIGS. 4A and 4B diagrammaticallyshow the model and FIGS. 4A-1 and 4B-1 show the expected results of LDcloning, whilst FIGS. 4A and 4A-1 show the model of the selective growthadvantage of rare progenitors in SCF/TGFα and oestradiol and FIGS. 4Band 4B-1 show the alternative model based on a change in thedifferentiation programme of many or all progenitors. If, according tothe first model, bone marrow contains both rare (one in 20,000) SCF/TGFαprogenitors with the ability to renew themselves and stable c-ErbBexpression, as well as additionally frequent (1 in 300) SCF progenitorswhich proliferate transiently in the presence of SCF but are capableneither of sustained self-renewal nor of expressing c-ErbB, SCF shouldinduce numerous proliferating clones of SCF progenitors after 4 to 6days. Thereafter, the number of proliferating clones should fall rapidlyon account of differentiating or degenerating SCF progenitors. In TGFαplus oestradiol, a much smaller number of clones (1 in 20,000) should beobtained which should remain substantially constant on account of thelong term self-renewal capacity of these clones. In the presence of allthree factors (SCF, TGFα and oestradiol) the numbers of colonies shouldinitially be as high as in SCF on its own but should thereafter fall tothe level obtained with TGFα plus oestradiol (FIGS. 4A-1). According tothe second model, bone marrow (and hence the SCF progenitors) containonly a few SCF/TGFα progenitors from the outset, whilst the majority ofthese cells develop from SCF progenitors in a slow process whichrequires the presence of SCF, TGFα, oestradiol (and chicken serumfactors). One would therefore expect the frequency of the clones whichdevelop in the presence of all three factors not to decrease in time orto decrease only slightly, which contrasts with the expected behaviourof such clones according to the first model (FIGS. 4B-1). The frequencyof the clones developing, on the one hand, in the presence of SCF and,on the other hand, in the presence of TGFα and oestradiol shouldcorrespond to that of the first model (FIGS. 4B-1).

a) LD cloning of purified SCF progenitors

Purified three day old SCF progenitors were prepared as described byHayman et al., 1993. The cells were then seeded in variousconcentrations (20 to 2,500 cells per well of the 96-well cell cultureplate) into CFU-E medium containing either oestrogen alone (control) orSCF alone (plus the oestradiol antagonist ICI 164384 in order tosuppress the oestradiol activity contained in the serum), or TGFα plusoestradiol or SCF, TGFα and oestradiol. To ensure good cloningefficiency 50 adherent myeloid cells were seeded into all the wells as afeed layer (the myeloid cells were obtained by preparing bone marrowcells and seeding them out at a rate of 50×10⁶ cells/ml per 100 mm dishand treating them with 10 ng/ml of cMGF and SCF. During the first 2 to 3days the non-adherent or only slightly adherent cells were extended andthen allowed to adhere in a larger dish.) Immature healthy colonies werecounted 4, 9 and 11 days after the seeding of the cells (correspondingto a total age of the cells of. 7, 12 and 14 days).

The results are shown in FIG. 5A (apart from the controls in FIG. 5,where very few colonies were obtained, the frequencies found are theresults of counting more than 100 colonies of at least two differentcell dilutions). As a control, first the total cloning efficiency(undifferentiated plus differentiated colonies) was determined, obtained2 to 3 days after seeding with the purified SCF progenitors in thevarious media (FIG. 5A, left panel). It will be seen that in thepresence of SCF cloning rates of 10 to 20% were obtained, irrespectiveof the presence of oestradiol or TGFα. In the media which contained TGFαplus oestradiol, or in the controls which contained only oestradiol, thefew visible colonies at this time were too small to be counted.

More conclusive were the results obtained with colonies which containedmore than 50% healthy, immature cells. On day 7, the number of clonescultivated in SCF alone had already fallen to <10⁻², whereas the clonescultivated in SCF, TGFα and oestradiol were still present in a frequencyof 10⁻¹. The frequency of the clones grown in TGFα plus oestradiol waseven smaller (2×10³), whereas the clones in the oestradiol controlsamples were not yet visible.

Further behaviour of the clones grown in the various media supported thehypothesis that SCF/TGFα progenitors develop from SCF progenitors.Immature clones growing in SCF alone fell to 3×10⁻⁴ or 1×10⁻⁴ after 12to 14 days, approximating to the background level (5×10⁻⁵) of thecolonies grown in oestradiol alone. As expected, the small number ofcolonies which had been grown in TGFα plus oestrogen (2×10⁻³) did notchange over time. In accordance with the finding that SCF progenitorscan develop into SCF/TGFα progenitors (FIGS. 4B and 4B-1) a considerableproportion of the clones grown in SCF, TGFα and oestradiol remainedimmature and capable of proliferation, whilst the frequency decreasedonly slightly (from 9×10⁻² on day 7 to 5×10⁻² on day 14; FIG. 5A).

b) LD cloning of normal bone marrow cells

To rule out that SCF progenitors with the ability to acquireself-renewing potential in SCF, TGFα and oestradiol had been preselectedby in vitro cloning before the LD cloning, tests were carried out withfresh untreated bone marrow cells to confirm the results obtained in a).In particular it was intended to determine whether erythroid progenitorswith self-renewing potential could indeed be generated from single cellsat frequencies approaching those of SCF progenitors (one progenitor in3,000-5,000; Hayman et al., 1993) if grown in all three factors, whilstremaining rare (1 in 15,000) if grown in TGFα plus oestradiol alone.

Normal bone marrow cells prepared as described by Hayman et al., 1993were seeded into CFU-E medium containing various factor combinations at4 different cell dilutions (500, 2,000, 6,000, 15,000) in a range offrom 500 to 15,000 cells per well, and immature colonies (containingmore than 50% round proliferating cells) were counted at various timesafter seeding. The results are shown in FIG. 5B: after 4 days, the cellsgrown in SCF formed colonies with a frequency of 3×10⁻² to 5×10⁻².Thereafter, the frequency of immature colonies decreased progressively,reaching a frequency of 2×10⁻⁵ after 13 days. An increasing proportionof cells underwent differentiation and then apoptosis. As expected, theclones grown in TGFα plus oestradiol were rare from the beginning(6×10⁻⁵ to 8×10⁻⁵) but the frequency remained substantially constantduring the experiment. On the other hand, the clones grown in SCF, TGFαplus oestradiol were found on day 4, 8 and 13 with a frequency of 3×10⁻²to 5×10⁻². Thus, the three factors SCF, TGFα and oestradiol can actuallyinduce the outgrowth of immature colonies from bone marrow with afrequency corresponding to that of SCF progenitors after 4 days andsimilar to the frequency of cells in normal chicken bone marrow whichare capable of forming CFU-E colonies. Finally, it was to be establishedwhether all three factors are actually necessary to induce the outgrowthof immature LD clones with a high frequency. In media containingindividual factors (oestradiol alone, TGFα or SCF plus ICI 164384 inorder to suppress the endogenous serum oestradiol) only very smallnumbers of immature clones were obtained (about 10-5). In TGFα plus SCFwithout oestradiol the clones behaved exactly as in SCF on its own, i.e.they were frequent on day 4 and then decreased progressively (FIG. 5B).Surprisingly, the clones cultivated in SCF plus oestradiol remainedimmature for much longer than those cultivated in SCF alone, but grewmuch more slowly compared with clones cultivated in TGFα plus oestrogenor SCF or in TGFα plus oestrogen. Since these clones bore no resemblanceto the typical SCF/TGFα progenitors (in terms of both c-ErbB expressionand in vitro lifespan, cf. Example 4), they were not investigatedfurther.

EXAMPLE 4

Investigation of the in vitro lifespan and expression of TGFαR/c-ErbB ofSCF/TGFα progenitors developed from SCF progenitors

In order to investigate whether the immature clones obtained by LDcloning of normal bone marrow cells or SCF progenitors in SCF, TGFα plusoestradiol with great frequency were actually typical SCF/TGFαprogenitors, they were examined both in terms of their in vitro lifespan and also their expression of TGFαR/c-ErbB and their proliferativeresponse to TGFα and other factors. The tests were carried out incomparison with cells grown only in TGFα plus oestradiol and also withcells from SCF/TGFα progenitor mass cultures.

a) Determining the life span

In order to analysis the in vitro lifespan, 10 to 12 healthy immaturecolonies cultivated in SCF, TGFα and oestradiol (obtained from 96 wellplates with 500 seeded out cells) or grown in TGFα plus oestradiol (fromplates with 15,000 cells) were isolated, suspended and expanded in theirrespective media until 20×10⁶ cells were obtained in a 100 mm dish orthe cells stopped growing because they had reached their clone specificin vitro lifespan (cell senescence). The growing clones were thenpassaged (diluted and transferred with fresh medium into new culturedishes) until they also aged. All immature colonies obtained after 13days' growth in SCF alone (6 colonies), as well as those from thecontrol cultures (oestradiol alone: 5 colonies; SCF alone: 5 colonies;TGFα alone: 3 colonies; SCF plus TGFα: 8 colonies; SCF plusoestradiol: >15 colonies) were treated similarly.

Clones which exhibited the lifespan predicted for the SCF/TGFαprogenitors were obtained only in SCF, TGFα plus oestradiol and, asexpected, in TGFα plus oestradiol. 8 out of 12 of the clones grown withhigh frequency in SCF, TGFα plus oestradiol had a lifespan of 23 to over28 generations (the remaining 4 had a lifespan of 12 to 15 generations).7 out of 10 clones which had grown in TGFα plus oestradiol with a lowerfrequency had a similarly high life expectancy (23 to 31 generations;the lifespan of the remaining 3 was 15 to 17 doublings). This clearlyshows that the life expectancy of SCF/TGFα progenitors which haddeveloped from SCF-progenitors in the presence of SCF, TGFα plusoestradiol, is identical to those of genuine SCF/TGFα progenitors. Noneof the colonies which had formed in the presence of individual factorsor SCF plus TGFα had a lifespan of more than 12 to 16 generations. Oneclone obtained in SCF plus oestradiol could be cultivated up to the 22ndgeneration whereas 9 others had a short lifespan (12 to 18 generations).However, this clone grew at reduced speed, expressed very small amountsof TGFαR/c-ErbB and did not react to TGFα in a growth factor assay. Itcan therefore be assumed that these cells are more likely to be anabnormal cell clone than actual SCF/TGFα progenitors.

b) Expression of TGFαxR/c-ErbB and response to TGFα and other growthfactors

To determine whether the LD clones obtained with high frequency in SCF,TGFα plus oestradiol express TGFαR/c-ErbB in similar amounts to SCF/TGFαprogenitors grown in TGFα plus oestradiol, all the factors were removedovernight from the cells of 5 LD clones (2 clones were combined becauseof the low number of cells) cultivated in all three factors, from 2clones cultivated in TGFα plus oestradiol and from an SCF/TGFαprogenitor mass culture, then the cells were lysed and investigated byWestern blot using anti-c-ErbB antibodies for TGFαR/c-ErbB expression.FIGS. 6A, and B (c-ErbB expression of LD clones from bone marrow)clearly shows that somewhat fluctuating but similar quantities ofTGFαR/c-ErbB were expressed in all three cell types, which againindicates that the erythroblast clones formed from SCF progenitors inthe presence of all three factors are genuine SCF/TGFα progenitors.

In order to determine more quantitively the extent to which the largenumber of LD clones obtained in the presence of the three factors issimilar to SCF/TGFα progenitors, another method was used: LD clonesinduced from purified SCF progenitors by cultivation in the threefactors (see FIG. 5B) were counted on day 13 and the plates on which themajority of wells contained an immature culture were selected. Then thecontents of all the wells were suspended, washed in medium withoutfactors and transferred into new 96 well plates containing mediumsupplemented with TGFα plus oestradiol. Control LD clones obtained fromTGFα plus oestradiol, SCF on its own and oestradiol on its own, weretreated similarly. 3 days later (day 16) the clones were investigatedfor their proliferative capacity by measuring the ³ H!thymidineincorporation (wells with a number of counts 5 times (for the individualcolonies) or 10 times (2 or more colonies) above the background levelwere counted as positive. From this analysis it was possible tocalculate the frequency of the thymidine-incorporating clones (FIG. 6B,panel B; the filled-in bars indicate the thymidine-incorporating clones;the shaded bars indicate all the clones). The data obtained show thatessentially all healthy immature clones which had been cultivated inTGFα plus oestradiol and identified on day 13 incorporated thymidine onday 16, confirming that they was still actively proliferating. The samewas true of more than 50% of the (30 times more numerous) clones whichhad been generated in the presence of all three factors. By contrast,fewer than 10% of the few clones which had survived after 13 days in SCFalone, incorporated thymidine, whereas the similarly rare clones whichhad grown out in the presence of oestradiol alone showed noproliferation whatever in TGFα plus oestradiol. This led one to assumethat the clones generated in the controls are not typical SCF/TGFαprogenitors, a finding which is confirmed by their short in vitrolifespan (see a)).

Finally, the intention was to confirm that the LD clones grown from SCFprogenitors at high frequency in the presence of all three factorsdemonstrate a similar dependency on SCF, TGFα and oestradiol to SCF/TGFαprogenitors. FIG. 6C, shows that an LD clone designated C6 (cf. FIG.6A,) showed a clear, concentration-dependent reaction to all threefactors, which nearly corresponded to the characteristics of an SCFprogenitor mass culture which had been cultivated for 20 days in thepresence of the three factors (see FIG. 3C) or SCF/TGFα progenitorscultivated in TGFα plus oestradiol alone.

EXAMPLE 5

Definition of the factors which are necessary to change thedifferentiation programme of SCF progenitors into SCF/TGFα progenitors

The results obtained in th e p receding Examples show that SCFprogenitors can develop into SCF/TGFα progenitors, i.e. they acquire thecapacity for both sustained self-renewal and also for the expression ofendogenous TGFαR/c-ErbB when cultivated in the presence of the threefactors. However, the fact that such cultures are critically dependenton the presence of chicken serum which may contain small concentrationsof TGFα, SCF and/or oestradiol as well as additional uncharacterisedfactors, set limits on the evaluation of the data and raised a number ofquestions. It remained unclear whether SCF/TGFα progenitors requiredsmall concentrations of SCF, which are certainly present in chickenserum. In addition, SCF progenitors could require small quantities of achicken factor which functionally replaces TGFα and which is alsocontained in chicken serum. Secondly, it was unclear at what time duringthe development of SCF/TGFα progenitors from SCF progenitors the variousfactors were required. And finally there was the question as to whichfactor or factors in chicken serum is or are required for theTGFα/oestradiol-induced outgrowth of SCF/TGFα progenitors from bonemarrow and whether this factor or these factors constitute a newactivity or a known factor, e.g: SCF.

In order to answer these questions it was necessary to prepare a batchof chicken serum which was substantially free from endogenous growthfactor and hormone activities but which still fully permitted the growthof factor-dependent cells when the necessary growth factors were addedfrom outside. Initial tests had shown that chicken serum treated withanimal charcoal (Schroeder et al., 1992) strongly inhibited theTGFα/oestradiol-induced outgrowth of SCF/TGFα progenitors (although didnot completely suppress it), but did not affect the growth rate of theseprogenitors once they were established. Therefore, chicken serum whichhad been thoroughly freed from endogenous hormones-and factors by freontreatment and subsequent three time treatment with animal charcoal(Schroeder et al., 1992) was used for the present experiments (thisdepleted serum is referred to hereinafter as "treated chicken serum".Bone marrow cells were cultivated in CFU-E medium containingfreon-treated foetal calf serum and either untreated chicken serum ortreated chicken serum. The cells were cultivated either in SCF alone orin SCF, TGFα and oestradiol and counted at the times specified in FIGS.7A-7C which shows the cumulative cell numbers determined as inExample 1. CFU-E medium prepared with the treated chicken serum (inFIGS. 7A-7C the open squares indicate purified chicken serum plus SCF;solid squares indicate purified chicken serum plus oestradiol; opencircles denote normal chicken serum plus SCF and solid circles indicatenormal chicken serum with oestradiol), made it possible for SCFprogenitors to grow to the same extent as the control medium withuntreated chicken serum, irrespective of whether the cells werecultivated in SCF alone or in SCF, TGFα plus oestradiol (FIG. 7A,).Moreover, there was no effect on the proliferation rate of 15 day oldSCF/TGFα progenitor cultures, apart from a slight effect when the cellsbegan to age (FIG. 7C). Surprisingly, however, the treated chicken serumslowed down the development of SCF progenitors into SCF/TGFα progenitorsin the presence of SCF, TGFα and oestradiol (FIG. 7B). After theirdelayed appearance, however, the SCF/TGFα progenitors generated intreated chicken serum grew at the same rate as the control cells inuntreated chicken serum which had formed at least 5 days earlier (FIG.7B).

These observations permit numerous conclusions: firstly, chicken serumcontains an additional activity which promotes the development of SCFprogenitors into SCF/TGFα progenitors. And secondly this activity isimportant for the switch in development, but does not affect theproliferation of SCF progenitors before the change nor is important forthe proliferation of already established SCF/TGFα progenitors. Theavailability of a suitably treated chicken serum also made it possibleto investigate at what time during the development of SCF/TGFαprogenitors the known factors are required. 3 day old purified SCFprogenitors grew at comparable speed in SCF plus TGFα, independently ofthe presence or absence of oestradiol (FIG. 8A, the oestradiol presentin the normal chicken serum used was again suppressed with ICI 164384).Thus, oestradiol has no effect on the early proliferation of SCFprogenitors. The fact that they grew at the same rate in mediacontaining treated chicken serum, SCF and oestradiol with or withoutTGFα shows that TGFα is also dispensable and that the only factorrequired by early SCF progenitors is SCF.

A different pattern of requirements of growth factors is produced duringthe change in the differentiation programme. As shown in FIG. 8A, cellskept in SCF plus TGFα without oestradiol irreversibly ceased toproliferate around day 8 to 10, indicating that oestradiol is necessaryfor the switch. Earlier results indicate that it is also essential forthe proliferation of established SCF/TGFα progenitors (Schroeder et al.,1993). Another group of experiments clearly shows that SCF is necessaryduring the change in the differentiation programme. 6 day old SCFprogenitors established in media containing treated chicken serum andSCF are able to develop with low efficiency into SCF/TGFα progenitors ifthey are further cultivated in treated chicken serum which contains allthree exogenous factors. However, if they are given only TGFα andoestradiol, under otherwise identical conditions, they lose this abilityentirely (FIG. 8B). Therefore, the development of SCF/TGFα progenitorsis dependent on the presence of SCF during the change in thedifferentiation programme, whilst, once established, these progenitorsare independent of SCF (see FIG. 7C, and below). Finally, SCFprogenitors do not require any TGFα (FIG. 7A,) but no formation ofSCF/TGFα progenitors occurs in the absence thereof, even if untreatedchicken serum is used (Schroeder et al., 1993). To sum up, the testscarried out lead to the following conclusion: the joint presence of SCF,TGFα and oestradiol is necessary for the development of SCF/TGFαprogenitors from SCF progenitors, whilst an unknown further activity inchicken serum increases the efficiency of their formation.

Some data from guide experiments which were additionally carried outlead one to suppose that this activity might be chicken erythropoietin,but they do not prove this. It was found that anaemic serum in growthfactor assays strongly stimulates the proliferation of SCF/TGFαprogenitors; a more important finding was the fact that anaemic serumincreased the growth rate of SCF/TGFα progenitors during and after theestablishment thereof, even when these cells were exposed to normalchicken serum plus SCF, TGFot and oestradiol ("STE") (FIG. 8C,).

Finally, erythroblasts which had been stimulated to self-renewal bymeans of a retrovirus stably expressed c-ErbB, i.e. an exogenoustyrosine kinase and which expressed the murine erythropoietin receptorafter infection with another retrovirus, were able to be stimulated intheir proliferation rate threefold or more by means of human recombinanterythropoietin (EPO).

EXAMPLE 6

Identification of two factors from chicken serum which speed up theconversion of SCF progenitors into SCF/TGFα progenitors or are necessaryfor their growth

a) Ligands of the glucocorticoid receptor (e.g. dexamethasone) belong tothe factors from chicken serum which SCF progenitors require for theirdevelopment into SCF/TGFα progenitors

The previous Example showed that the development of SCF progenitors intoSCF/TGFα progenitors required, in addition to SCF, TGFα and oestradiol,other undefined factors from chicken serum which could be eliminated byactivated charcoal treatment of the serum. In the presence of a chicken'serum treated with activated charcoal, the development into SCF/TGFαprogenitors does not occur or takes place very inefficiently.

Since steroid hormones in particular are eliminated by activatedcharcoal treatment of serum, other steroid hormones apart fromoestradiol were investigated for their activity during the conversion ofthe differentiation programme of normal erythroid cells. First of all,ligands of the glucocorticoid receptor were tested, since a deficiencyof glucocorticoids in humans lead inter alia to anaemia and prevents theDMSO-induced differentiation in Friend-erythroleukaemia cells in themouse. Preliminary experiments showed 1) that SCF cells do not requireDMSO for their temporary self-renewal and 2) established SCF/TGFα cellsrequire small concentrations of glucocorticoids for their growth. Thecells do not grow when cultivated in the presence of TGFα and oestradiolin media in which both the foetal calf serum and the chicken serum havebeen treated with activated charcoal. If an additional 1×10⁻⁶ Mdexamethasone is added to the same medium, the cells are stimulated togrow at normal speed. The cells may also fail to grow even in untreatedmedia if a glucocorticoid antagonist is added as well as the TGFα andoestradiol.

In order to test directly whether glucocorticoids (dexamethasone) speedup the conversion of SCF into SCF/TGFα progenitors, an experiment wascarried out in which SCF progenitors were exposed to various factormixtures for a short time (4 days, day 3-7 after isolation of the bonemarrow, hereinafter referred to as the induction period) (see FIGS. 9Aand 9B). The cells were then washed and seeded into medium containingonly TGFα and oestradiol (TE medium). In this medium, only c-ErbBexpressing, fully developed SCF/TGFα progenitors can grow, but not SCFprogenitors or cells at an early stage of development into SCF/TGFαprogenitors (see Example 5). The results are shown in FIGS. 9A and 9B.

As a negative control, the cells (4 day old SCF progenitors)werecultivated during the induction period in SCF plus medium with activatedcharcoal-treated sera (foetal calf serum and chicken serum). Afterswitching to TE medium, no cell growth could be observed for a longtime. Not until 9-10 days after the switch to TE medium had passed didSCF/TGFα progenitors grow out (FIGS. 9A and 9B, white lozenges) whichpresumably derived from cells already present in the bone marrow asSCF/TGFα progenitors (see Example 5).

As a positive control, the SCF progenitors were treated during the 4-dayinduction period with SCF, TGFα and oestradiol (FIG. 9A, blacktriangles). After transfer into TE medium the cells grew out very muchfaster, as expected, and there was only a 5 day delay (lag-phase) inoutgrowth. This corresponds to the results shown in Example 1 (FIG. 1,arrows).

A surprising result was obtained when the cells were treated with SCF,TGFα, oestradiol and dexamethasone during the induction period. Not onlydid the cells grow much faster during the induction period than in thecontrols, but also after transfer into TE medium there was noappreciable lag phase--the cells carried on growing at a constant speed(FIG. 9A, white squares). This result shows that the addition ofdexamethasone brings about the conversion of virtually all SCFprogenitors into SCF/TGFα progenitors. Additional investigations usingphosphotyrosine blot (Western blot with phosphotyrosine antibodies)showed that these cells expressed the expected quantities of c-ErbB.

The effect of dexamethasone could also be observed in cells grown onlyin the presence of SCF. The addition of dexamethasone in the presence ofSCF speeded up the outgrowth of SCF/TGFα progenitors more strongly thanin the positive control (SCF, TGFα, oestradiol, FIG. 9A, cf. black andwhite triangles. As in Example 5, it was apparent that the cellsrequired the oestradiol contained in small amounts in the sera, inaddition to SCF and dexamethasone, since the addition of the oestradiolantagonist designated ICI 164384 (Schroeder et al., 1993) limited theoutgrowth of the cells to the extent observed in the negative control(FIG. 9A, cf. white lozenges and black circles). In addition, the cellsrequired small concentrations of a c-ErbB ligand (unknown, contained inchicken serum).

These results show i) that dexamethasone is necessary, in addition toTGFα and oestradiol, for the growth of SCF/TGFα progenitors capable ofself-renewal and ii) that this hormone greater accelerates theconversion of SCF progenitors into SCF/TGFα progenitors.

b) Growth of SCF/TGFα progenitors: insulin-like growth factor I (IGF-1)together with SCF, TGFα, oestradiol and dexamethasone replaces thechicken serum which is absolutely essential for cell growth.

All previous experiments with normal erythroid chicken precursor cellscapable of self-renewal were linked to the presence of tested batches ofchicken serum; it has not yet been possible to define all the factorscapable of replacing chicken serum. The results described in a), namelythat dexamethasone permits the growth of these cells in chicken seratreated with activated charcoal, led to a series of attempts to replacethe chicken serum with defined factors. The definition of these factorsnecessary for chicken cells forms the basis for any correspondingrequirements of human cells.

Since a factor mixture of SCF, TGFα, oestradiol and dexamethasone wasideal for promoting the development as well as the growth of SCF/TGFαprogenitors, this mixture was used in media with and without chickensera. As possible other factors for replacing chicken serum,insulin-like growth factor (IGF-1) and avian IL-6 (ChickenMyelomonocytic Growth Factor, cMGF) were investigated. The experimentswere carried out in medium with (FIG. 9B, S13 medium) and withoutchicken serum (FIG. 9B, Epotest).

Of the factors tested, only IGF-1 was effective. FIG. 9B shows that inthe presence of SCF (S), TGFα (T), oestradiol (E), dexamethasone (D) andIGF-1 (IG), both 16 day old SCF/TGFα progenitor cells (FIG. 9B, blackcircles, white triangles) and 9 day old bone marrow cells cultivated inSCF/TGFα and oestradiol (FIG. 9B, black and white triangles)proliferated equally rapidly in media with chicken serum (black symbols)and without chicken serum (white symbols). The effect could be detectedover a period of >7 days. In the absence of IGF-1 the cells stoppedgrowing completely after 2 days. The same results (no cell growth) wereobtained when IGF-1 was replaced by cMGF.

EXAMPLE 7

Cultivation of human erythroid cells resembling the chicken SCF andchicken SCF/TGFα progenitors

a) Provisional definition of conditions which make it possible for humanerythroid progenitors to grow out of bone marrow or peripheral blood

Experiments were carried out using human haematopoietic cells. Theassumption underlying these experiments was that human erythroidprogenitors have an in vitro lifespan similar to that of humanfibroblasts (50 to 70 generations), and this constitutes the basis fordetecting human erythroid progenitors capable of self-renewal.

Either bone marrow or peripheral blood from healthy donors served as thesource for these experiments. From these sources, immature blood cellswhich express the CD34 cell surface antigen were concentrated usingimmune affinity chromatography as described by Shpall et al., 1994. Theconcentrated cells were seeded, as in the previous Examples, into amodified CFU-E medium (Hayman et al., 1993) containing human serum(Sigma) instead of chicken serum and iron-saturated human transferrin(Sigma) instead of conalbumin. The medium was supplemented with 20 ngTGFα (Promega), 20 ng recombinant EGF (Promega EGF was used in case thehypothetical member of the EGF receptor family present in erythroidcells does not have TGFα as a functional ligand), 100 ng of purifiedhuman SCF (Promega), 5×10⁻⁷ M oestradiol (in some experiments whichserved to characterise the cells other factors such as IL-3, IL-1 andLIF were added to the medium). The cell growth was monitored by cellcounting and the cell types present in the cultures were analysed bycytocentrifugation on slides and histochemical staining on haemoglobinand histological dyes (Beug et al., 1982).

i) Experiments with bone marrow

The initial attempts to grow erythroid progenitors from human bonemarrow in modified CFU-E medium containing human serum, iron saturatedhuman transferrin, 20 ng TGFα (Promega), 20 ng recombinant EGF(Promega), 100 ng of purified human SCF (Promega), 5 ×10⁻⁷ M oestradioland various other factors (10 ng each of IL-3, IL-6, IL-1 and LIF) permillilitre (ml), were initially unsuccessful. However, when recombinantEPO (3 International units/ml) was added to the medium, erythroidprogenitors could be grown out, which remained immature for 13 days butwhich were substantially all differentiated on day 16. During this timethe numbers of cells increased 25 to 50 fold; a more accuratemeasurement was impossible owing to the low cell numbers (only 2×10⁶ ofcells initially seeded out, therefore fewer than 10⁵ cells after 3-5days). The proliferating cells obtained resembled humanproerythroblasts, and surprisingly they were similar to the normalerythroid chicken progenitor cells (FIGS. 11A-1, 11A-2 and 11B, seebelow). During the first few days of the culture, and also after day 15,numerous nucleus-containing reticulocytes, nucleus-expelling cells anderythrocytes were visible, indicating that the differentiatingreticulocytes in the culture normally differentiated to becomeerythrocytes and were also normally able to carry out the process ofenucleation (ejection of the nucleus). Without EPO the cultures did notgrow and contained very few immature erythroid cells. They containedmainly maturing monoblasts and various types of immature granulocytes(neutrophiles, eosinophiles, mast cells).

ii) Experiments with cells from peripheral blood The experimentdescribed in i) was repeated with 40×10⁶ CD34⁺ cells, concentrated fromhuman peripheral blood. 2×10⁶ cells/ml in modified CFU-E medium plusSCF, TGFα and EGF, oestradiol and human recombinant EPO were seeded intotissue culture dishes and the cells were counted at the times specified,the average cell volumes being determined in an electronic cell counterof the CASY-1 type, Sharp system. Since the initial number of cells wasgreater than in the experiment carried out with bone marrow, theproliferation kinetics of the culture could be monitored accurately.FIG. 10A shows that the cell numbers decreased during the first 2 to 3days, which can be put down to the maturation and/or cell death ofpartially differentiated progenitors. Subsequently, the cellsproliferated exponentially with doubling times of between 20 and 30hours up to day 15, after which no further growth was observed. Thetotal increase in cell numbers during this growth phase was >300 fold.FIG. 10A also shows that during the phase of exponential growth thecells maintained their size (cell diameter between 9 and 10 μm, cellvolumes between 500 and 600 femtolitres), which is a first indicationthat they remained immature.

Since antigenic markers which distinguish human proerythroblasts fromother myeloid or multipotent progenitors were not available for carryingout these experiments, and detection by histological staining is nottruly definitive, indirect methods were used for determining thepercentage of erythroid progenitors in the cultures: first, aliquotswere stained at regular intervals of time using acid benzidine, which isa very sensitive haemoglobin detector (Graf and Beug, 1978). On the 6thday the cultures already contained 14% benzidine-positive cells and ondays 10 and 11 these levels had risen to 51 and 63%. Since a pureculture of chicken SCF/TGFα progenitors contains between 30 and 60%benzidine-positive cells, these results indicate that on about day 10the culture consisted predominantly of erythroid progenitors. This viewcould be confirmed by a test in which the cells were induced todifferentiate: 1 aliquot of the 10 day old culture was washed andresuspended in modified CFU-E medium containing 10 units/ml of humanrecombinant EPO plus 10 ng/ml of insulin or IGF-1 (insulin like growthfactor 1). A parallel aliquot was additionally given IL-3 (10 ng/ml).The data in FIG. 10B show that the cell numbers increased approximately3-fold, whilst the cell volume decreased significantly at the same time,as is to be expected for differentiating erythroid cells. An acidbenzidine staining carried out after 2 days yielded more than 95%benzidine-positive cells in the culture which had been given EPO/insulinalone. This indicates that the majority of the cells present before theinduction of differentiation must have been erythroid, particularly asvery few apoptotic cells were visible in the differentiating culturesafter cytocentrifugation and histological staining (see below). Theaddition of IL-3 probably delayed differentiation; after 2 days, only66% benzidine-positive cells were detected, and the cells grew somewhatfaster, whilst their cell volume decreased more slowly (FIG. 10B).

b) Characterisation of the cells proliferating in SCF, TGFα, oestradioland EPO

In order to determine whether the erythroid progenitors obtained byculture in SCF, TGFα plus EGF, oestradiol and recombinant EPO correspondto the chicken SCF/TGFα progenitors grown in the preceding Examples, thefollowing two test methods were used:

Firstly, the cell types present in the cultures were characterised bycentrifugation on slides and combined histological and histochemicalstaining for haemoglobin (Beug et al., 1992, see the stages definedtherein). The intention was to determine how long immaturehaemoglobin-negative or slightly positive proerythroblasts would last inthe cultures, so as to obtain some indication as to whether theerythroid progenitors actually underwent self-renewal, as was to besupposed on the basis of growth kinetics and size distribution (FIG.10A). Proerythroblasts differ from other cells; in the staining used, bya central large cell nucleus, strongly basophilic cytoplasm,characteristic lapping of the cytoplasm seam and a slight staining withneutral benzidine which is distinguishable from myeloid cells. FIG. 11A,panel A (proliferating cells, bone marrow after 7 days, CD34⁺ cellsafter 10 days) and FIG. 11B (differentiated after 10 days proliferationand 4 days differentiation) shows that a large percentage of the cellswhich last in the culture resemble benzidine-negative protoerythroblastsand in addition there was some myeloid cells. These results wereobtained up till day 14, and then the percentage of maturing cellsincreased significantly. By contrast, the cells obtained after 4 days ofdifferentiation induction (see above) constituted recticulocytes and,nucleus-expelling and mature erythrocytes (FIGS. 11A-1, 11A-2 and 11B),which is further confirmation that the cells kept in SCF, TGFα ,oestradiol and EPO were actually prevented from entering into thedifferentiation induced by the above factors. FIG. 11A showspreparations of human bone marrow (BM) and CD34⁺ cells (CD34),characterised by centrifugation on slides and combined histological andhistochemical staining for haemoglobin, these preparations having beenphotographed under green light (above) and blue light (below) in orderto pick up any histological details and haemoglobin staining.Er=erythrocytes and nucleus-expelling erythrocytes; R=reticulocytes;Pe=proerythroblasts; M=myeloid cells. FIG. 11B, panel B shows CD34⁺cells cultivated for 10 days, which were induced to differentiate for 4days and photographed in a similar manner.

A clearer indication that cells resembling SCF/TGFα progenitors canactually be obtained from human erythroid progenitors was obtained byinvestigating whether the cells express both c-Kit and also a member ofthe c-ErbB/EGF receptor family and proliferate in response to theparticular ligands. Since it was initially impossible to obtain anycultures which underwent self-renewal during the expected 50 to 70divisions, it was thought that the majority of the cells in thecultures, particularly at early stages, correspond to SCF progenitorsand that SCF/TGFα progenitors were only generated with low efficiency,presumably as a result of sub-optimal culture conditions. The reactivityof the human bone marrow cells to various growth factors was thereforetested using various growth factor assays (Leutz et al., 1984; Hayman etal., 1993). The results of these assays are shown in FIG. 11C(self-renewal factors TGFα/EGF, SCF) and FIG. 11D (differentiationfactors (EPO, IL-3)). For these experiments,. CD34⁺ cells were grown for8 days, washed and tested for their growth factor dependency asdescribed in Example 4, except that CFU-E medium was used without humanserum. A relative growth factor concentration of 100 corresponded to 400ng/ml of recombinant SCF, and 40 ng/ml each of TGFα or EGF; 10 ng/ml ofhuman recombinant IL-1, 20 units/ml of human recombinant EPO, 40 ng/mlof human recombinant IL-3 and 10 ng/ml of recombinant murine LIF. Thevalues shown are the averages from three measurements. The cellsexhibited a strong reaction to SCF, and, more significantly, a weak butdistinct reaction to a mixture of TGFα and EGF. On the other hand, therewas no reaction to the two cytokines IL-1 and LIF, which act on veryearly, multi-potent haematopoietic cells. This leads one to concludethat the cells reacting to SCF are committed erythroid progenitors. Asexpected, the cells reacted just as strongly to the erythroiddifferentiation factors EPO and IL-3, again confirming that the culturepredominantly contains erythroid cells.

EXAMPLE 8

Additional growth factors and steroid hormones induce self-renewal forlong periods (>20 generations) in cultures of human proerythroblastscapable of terminal differentiation.

In Example 6, in the chicken system, results were obtained which were ofpotential significance for the outgrowth of human proerythroblastscapable of self-renewal:

1. Of the initially undefined factors in the chicken system which arenecessary in addition to SCF, TGFα and oestradiol for the development ofSCF progenitors into SCF/TGFα progenitors, two factors could beidentified: the steroid hormone dexamethasone and the general growthfactor insulin-like growth factor (IGF)-1.

2. The effect of dexamethasone on the self-renewal characteristics ofhuman proerythroblasts was therefore investigated more thoroughly.Similarly, investigations were carried out to discover whether IGF-1,which makes the cells independent of chicken serum in the chickensystem, shows at least growth-promoting properties on human cells. Theresults obtained were surprising: it was possible to increase thereplication of human proerythroblasts during their in vitro lifespanfrom 200-1,000 fold to more than 100,000 fold. This allowed moreaccurate characterisation of the cell populations obtained by colonytests and FACS analysis. In addition, the differentiationcharacteristics of the proerythroblasts could be investigated much moreaccurately than in Example 7 because there were sufficient cellsavailable.

a) Effect of dexamethasone

Human CD34⁺ cells from umbilical cord blood, purified as described inExample 7, part (ii)l w ere seeded into medium plus Epo, huSCF, TGFα andoestradiol, as described in Example 7. In addition to the above factorstermed the "factor mix" 1×10⁶ M dexamethasone was added to a secondculture. The cell growth was monitored until any detectable replicationhad ceased. The results are shown in FIG. 12A. As expected, cells growexponentially in "factor mix" up till day 13/14 and showed a 1,000-2,000fold cell multiplication (FIG. 12A, black circles). Unexpectedly, theparallel culture which was cultivated in factor mix plus dexamethasoneproliferated exponentially until at least day 18 and thereafter itsgrowth stopped only gradually (FIG. 12A, white squares), so as to obtaina 150,000 fold cell multiplication. If one assumes that some of thecells will always enter into spontaneous differentiation and hence onlysome of the immature cell population is available for maintaining theself-renewal potential of the culture, these data show that theproerythroblasts from human umbilical cord blood in the presence of EPO,SCF, TGFα, oestrogen and dexamethasone are capable of maintaining theirself-renewal potential for at least 20 cell generations. This issubstantially more than even the 7-10 cell divisions which human BFU-Esundergo within their normal development potential (Sawada et al., 1990).It is thus demonstrated i) that human erythroid progenitor cells,similarly to the corresponding cells in the chicken, can be induced toundergo a genuine change in their developmental potential, i.e. tosustained self-renewal, by combinations of tyrosine kinase ligands andligands of steroid receptors.

b) Effect of IGF-1

CD34⁺ cells from peripheral blood and an adult (obtained as in Example7, part ii) were cultivated in "factor mix" plus dexamethasone until thecells began to grow exponentially. Then an additional 40 ng/ml of humanrecombinant IGF-1 (Promega) were added to one aliquot of the cells. Asshown in FIG. 12B, the cells with IGF-1 (black circles) grewsignificantly faster than without this growth factor (white squares).Since the medium used contained 15% foetal calf serum and 4% serum fromhuman umbilical cord blood (which can be expected to contain a basalconcentration of IGF-1), an admittedly relatively small increase in thegrowth rate was achieved but it nevertheless shows that the cells reactto IGF-1. This is also apparent from experiments in which the cells werecultivated overnight without factor, then stimulated for 5 and 10minutes with IGF-1, lysed and investigated for receptor phosphorylationin the phosphotyrosine blot (see Example 2). The cells thus treatedexhibited autophosphorylation of the intracellular 90 kD IGF-1-receptor-chain and the 130 kD IRS-1 (insulin receptor substrate)protein, in the phosphotyrosine blot.

c) More accurate characterisation of the human erythroblasts capable ofself-renewal, by means of colony tests and surface markers

The surprising ability of the human proerythroblasts cultivated in"factor mix" with and without dexamethasone made it seem sensible toinvestigate these cells more closely for their developmental potentialand their position within the erythroid developmental series. Two typesof method were used for this:

Firstly, the cells were seeded into semi-liquid medium with suitablecombinations of cytokines and 10 days later the type of colonies grownwere counted. The following colony types were distinguished: i) burstforming unit erythroids (BFU-E), colonies consisting of 1,000 to >20,000cells, which contain only erythrocytes and thus show that the startingcell is an immature progenitor which is committed to the erythroidlineage; ii) BFU mix, large colonies with >20,000 cells, which contain,apart from erythrocytes, cells of at least one other lineage and thusindicate multipotent starting cells; and iii) colony forming unitsgranulocyte/macrophage (CFU-GM), colonies of 100 to >1,000 cells, whichcontain no erythrocytes, only myeloid cells (macrophages and/orgranulocytes) and thus derive from non-erythroid progenitor cells.

Secondly, the cells were tested using suitable antibodies and FACSanalysis for the expression of surface markers which are specific tocells of certain lineages and degrees of maturity. Although there are nosurface markers in existence which when used individually will recogniseexclusively human proerythroblasts, by combining a number of markerswhich are expressed on immature and/or mature erythroblasts withspecific markers for myeloid cells (CD 33) and lymphoid cells (CD-3,CD-19) it is possible to determine with great certainty whether thecells belong to the erythroid lineage. The CD 71 (α-transferrinreceptor) antibody is particularly suitable for this, as it admittedlystains all proliferating cells slightly but marks erythroid cells verystrongly. Together with CD 117 (c-Kit, expressed only on totallyimmature, BFU-E-like erythroid cells and multipotent precursors andcertain myeloid cells (mast cells)) and GPA (α-glycophorin, specific topartially mature erythroid cells), the CD-71 antibody makes it possiblesafely to determine proerythroblasts (CD 71 bright, CD 107 positive toslightly positive, GPA negative or slightly positive, CD33, CD3, CD 19negative).

In order to test cells from the corresponding cultures for theirmembership of a lineage and their degree of maturity within theerythroid lineage, cell aliquots were taken from the cultures shown inFIG. 12A on day 13 and day 16 and subjected to the tests shown in TablesI to III. The cells taken from the culture without dexamethasone on day16 were also separated according to density: in the chicken system, onlycells with a density of <1.070 g/cm³ are immature, cells with a densityof >1,072 g/cm³ are in all cases partially mature and required only 1-2days to mature into erythrocytes, during which time they underwent onlya few cell divisions. Corresponding fractions were prepared from thehuman cells and were tested separately for colony formation and surfacemarkers.

The results are shown in Tables I to III. These show that thepredominant type of colony is formed after both 13 and 16 days fromBFU-Es, i.e. from immature erythroid progenitors. The effect ofdexamethasone to keep the cells for longer in an immature statecharacterised by self-renewal is also made clear by the BFU-E numbers:the cultures with dexamethasone contain 2-2.5 times the number ofimmature colony-forming erythroid progenitors as without dexamethasoneafter both 13 and 16 days. The data also show that the cell populationconsists overwhelmingly of committed erythroid progenitors. After both13 and 16 days, more than 90% of the colony-forming cells are purelyerythroid progenitors and the proportion of multipotent (BFU-mix) andmyeloid "committed" progenitors is only 3-6%. Interestingly,dexamethasone also stimulates the content of multipotent progenitors 3-6fold, whereas the effect on the myeloid progenitors is much weaker.

As expected, the denser fraction (>1.072 g/cm³) of 16 day old umbilicalcord blood cells was incapable of forming colonies in semi-liquidmethocel medium. Its more mature nature was also confirmed by markeranalysis.

Tables II and III also show that the results of the marker analysis inthe FACS fully confirmed the conclusions drawn from the colony tests.All the cultures contained only a small amount of myeloid cells (10-20%after 13 days, 5-7% after 16 days, no lymphoid cells and scarcely anyCD34 positive cells (around 5%) data not show). Around 85% of the cellsare strongly CD71 positive but only a few percent of the cells are GPApositive. The effect of dexamethasone of keeping the progenitor cells inan immature state is also clarified by the CD 107 (c-Kit) expression: onday 16 (when the culture without dexamethasone was clearly decreasing inspeed of growth; see FIG. 12A) only 21% of the cells are c-Kit positive,whereas in the exponentially growing parallel culture kept in thepresence of dexamethasone, more than 50% of the cells were c-Kitpositive. The partially mature state of the denser fraction from the 16day old culture was confirmed by marker analysis: only 53% of the cellswere CD71 positive (maturing cells lose the transferrin receptor)whereas 66% of the cells were GPA positive.

To summarise, characterisation of the cultures from umbilical cord bloodshowed the following:

i) The cultures consisted predominantly of immature,proerythroblast-like progenitors which are committed to the erythroidlineage but can still form large erythroid colonies (BFU-E).Contamination with multipotent cells and cells of other lineages makesup less than 10%.

ii) The effect of dexamethasone, together with other factors, of-inducing the capacity of human proerythroblasts for sustainedself-renewal (more than 16 cell divisions) is clearly reflected inanalysis of the colonies and markers: both the capacity to form BFU-E(and BFU-E mix) and the capacity to express c-Kit are critically boostedby dexamethasone.

d) Regulation of the differentiation of in vitro cultivatedproerythroblasts from umbilical cord blood: application of the resultsobtained in the chicken system.

A major advantage of normal erythroid progenitor cells capable ofself-renewal in the chicken system was that after the removal of "selfrenewal factors" (SCF, TGFα and oestradiol) and replacement of thesefactors with differentiation factors (Epo, insulin) the cellsdifferentiated out with normal kinetics and undergoing the expectednumber of cell divisions (Hayman et al., 1993). Example 7 has alreadyshown that this observation could be applied in principle to humanerythroblasts: human proerythroblasts cultivated in vitro matured inrecombinant human Epo and insulin into enucleating (nucleus-expelling)erythrocytes (Example 7, FIGS. 10A and 10B). The presence of largeramounts of human proerythroblasts clearly capable of self-renewal madeit possible to investigate this differentiation inductionquantitatively. Moreover, it was now possible to analyse the effect offurther factors on the differentiation programme of the cells. In thechicken system it was possible to show that SCF in the presence of Epoand insulin was able to significantly delay erythroid differentiation orvirtually prevent it during the first 4-5 days (Hayman et al., 1993).Moreover, the thyroid hormone T3 (triodo-tyronine), particularlytogether with ligands of the co-receptor RXR, was capable of speeding upthe erythroid differentiation and completely reversing the slowing downof the erythroid differentiation caused by SCF (Schroeder et al., 1992;Beug et al., 1994). It was therefore of interest to discover whetherthese observations made with regard to the chicken system were alsovalid for the human system. Whereas a significant effect of SCF, whichmight be interpreted as a delay in differentiation, on purified humanBFU-E could be detected (Dai et al., 1991; Sawada et al., 1991) thereare no known direct investigations into the effect of T3 on thedevelopment of purified erythroid progenitors. The experiments werecarried out with 16 day old cells from the culture kept with "factormix" and dexamethasone. The cells were centrifuged, washed in mediumwithout factors and cultivated in a density of 1-2×10⁶ cells/ml in thevarious differentiating media. The differentiating medium contains 2%human serum (from umbilical cord blood) and either no further additives(FIG. 13, white squares; no factor), 10 units/ml of human recombinantEpo plus 10 ng/ml of insulin (FIG. 13, Epo, Ins; black squares), Epo,Ins plus 100 ng of human SCF (FIG. 13, SCF, Epo, Ins; white circles) andthe above factors plus 200 nm triiodothyronine and 10⁻⁶ M 9 cisretinolic acid (FIG. 13, SCF, Epo, Ins, T3, RXR Lig.; black circles).During the differentiation the cells were cultivated at a density of2-4×10⁶ cells/ml and fresh factors were added daily. At the timesspecified the cell volume was determined in an electronic cell counterof the type CASY-1, sharp system (see Example 7). At the same time, thehaemoglobin content of cell aliquots of known cell number was determinedby photometric measurement (Kowenz et al., 1987). The results are shownin FIG. 13. Whereas the haemoglobin content/cell volume scarcelyincreased in the absence of Epo/Ins (FIG. 13, white squares), in thepresence of Epo/insulin there was a sharp (approximately 8 fold)increase in the haemoglobin content/cell volume (FIG. 13, blacksquares). Surprisingly, SCF delayed the erythroid differentiationinduced by Epo/insulin just as in the chicken system (FIG. 13, whitecircles), whereas the addition of thyroid hormone (T3) plus RXR ligandcompletely reversed this delay in differentiation by SCF (FIG. 13, blackcircles), again exactly analogously to the data obtained in the chickensystem.

To summarise, these data show that human proerythroblasts capable ofself-renewal in culture mature, dependent on erythropoietin, in cultureinto mature erythrocytes which accumulate haemoglobin. This process isdelayed by SCF and accelerated by T3 (as in purified human BFU-Es,Sawada et al., 1991).

                  TABLE I    ______________________________________           Colony type           (per 10.sup.5 cells)    Cell type             BFU-E        BFU-mix  CFU-GM    ______________________________________    1        2250          60       70    2        5750         400      200    3        1200          45      115    4         <1           <1       <1    5        2500         105      100    6        ND           ND       ND    ______________________________________     ND = not detected     1: Umbilical cord blood (13 days, factor mix)     2: Umbilical cord blood (13 days, factor mix + dexamethasone)     3: Umbilical cord blood (16 days, factor mix, immature fraction <1.070     g/cm.sup.3)     4: Umbilical cord blood (16 days, factor mix, mature fraction >1.072     g/cm.sup.3)     5: Umbilical cord blood (16 days, factor mix + dexamethasone)     6: Peripheral blood (CD34.sup.+, 9 days, factor mix + dexamethasone)

                  TABLE II    ______________________________________           Cell surface marker           immature             CD71          CD117     erythroid             (Transferrin  (c-Kit, SCF-                                     GPA    Cell type             receptor)     receptor) (glycophorin)    ______________________________________    1        ND            ND        66%    2        ND            ND        20%    3        88%           21%        9%    4        53%           21%       66%    5        84%           51%        7%    6        80%           65%       30%    ______________________________________     ND = not detected     1: Umbilical cord blood (13 days, factor mix)     2: Umbilical cord blood (13 days, factor mix + dexamethasone)     3: Umbilical cord blood (16 days, factor mix, immature fraction <1.070     g/cm.sup.3)     4: Umbilical cord blood (16 days, factor mix, mature fraction >1.072     g/cm.sup.3)     5: Umbilical cord blood (16 days, factor mix + dexamethasone)     6: Peripheral blood (CD34.sup.+, 9 days, factor mix + dexamethasone)

                  TABLE III    ______________________________________              Cell surface marker                non-erythroid                           CD2, CD19    Cell type   CD22 (gran.)                           (B-cells, T-cells)    ______________________________________    1           10%        <0.1%    2           20%        <0.1%    3           5%         <0.1%    4           <0.1%      <0.1%    5           7%         <0.1%    6           5%         ND    ______________________________________     ND = not detected     gran. = granulocyte cells     1: Umbilical cord blood (13 days, factor mix)     2: Umbilical cord blood (13 days, factor mix + dexamethasone)     3: Umbilical cord blood (16 days, factor mix, immature fraction <1.070     g/cm.sup.3)     4: Umbilical cord blood (16 days, factor mix, mature fraction >1.072     g/cm.sup.3)     5: Umbilical cord blood (16 days, factor mix + dexamethasone)     6: Peripheral blood (CD34.sup.+, 9 days, factor mix + dexamethasone)

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We claim:
 1. A process for in vitro expansion of non-immortalizedhematopoietic CD34⁺ progenitor cells of erythroid lineage withoutsimultaneous differentiation, comprising(a) growing erythroid CD34⁺progenitor cells in a medium suitable for erythroid cell growth; and (b)exposing said cells to a combination of growth factors comprising(i) atleast one estrogen receptor ligand; (ii) at least one glucocorticoidreceptor ligand; and (iii) at least one tyrosine kinase receptorligand;for a time sufficient to induce said progenitor cells to beginself-renewal and thereby expansion, without simultaneousdifferentiation.
 2. The process of claim 1, wherein subsequent to step(b), said cells are exposed to at least one factor required forsustained self-renewal.
 3. The process of claim 2, wherein said CD34⁺progenitor cells are human cells.
 4. The process of claim 3, whereinsaid erythroid CD34⁺ progenitor cells are bone marrow cells, peripheralblood cells, or umbilical cord blood cells.
 5. The process of claim 1,wherein at least two tyrosine kinase receptor ligands are employed instep (b).
 6. The process of claim 5, wherein said two tyrosine kinasereceptor ligands bind to different classes of tyrosine kinase receptors.7. The process of claim 6, wherein said different classes of kinasereceptors differ with respect to their kinase domain.
 8. The process ofclaim 7, wherein one tyrosine kinase receptor ligand binds to a tyrosinekinase receptor selected from the sub-class of tyrosine kinase receptorshaving an uninterrupted tyrosine kinase domain, and a second tyrosinekinase receptor ligand binds to a tyrosine kinase receptor selected fromthe sub-class of tyrosine kinase receptors having a kinase insertionsequence.
 9. The process of claim 5, wherein said combination of growthfactors comprises(i) a ligand of c-Kit; and (ii) at least one ligand ofa receptor selected from the group consisting of the epidermal growthfactor family of receptors and the hepatocyte growth factor family ofreceptors.
 10. The process of claim 5, wherein said combination ofgrowth factors comprises:(i) stem cell factor; (ii) estradiol; (iii) atleast one of dexamethasone and hydrocortisone; and (iv) at least one oftransforming growth factor α, epidermal growth factor and hepatocytegrowth factor.
 11. The process of claim 5, wherein in step (b), saidcombination of growth factors further comprises at least one otherfactor capable of accelerating the initiation of self-renewal of saidcells, and wherein said other factor is a cytokine, a ligand of atyrosine kinase receptor, or a ligand of a serine kinase receptor. 12.The process of claim 11, wherein said other factor is erythropoietin.13. The process of claim 11, wherein said other factor is insulin-likegrowth factor
 1. 14. The process of claim 11, wherein said combinationof growth factors comprises stem cell factor, dexamethasone, estradiol,erythropoietin, insulin-like growth factor 1, and at least one oftransforming growth factor α, epidermal growth factor and hepatocytegrowth factor.
 15. The process of claim 11, wherein subsequent to step(b), said cells are exposed to at least one factor required forsustained self-renewal.
 16. The process of claim 15, wherein said factorrequired for sustained self-renewal is selected from the groupconsisting of a ligand of the family of epidermal growth factorreceptors, a ligand of the family of hepatocyte growth factor receptors,stem cell factor, erythropoietin, and insulin-like growth factor
 1. 17.The process of claim 15, wherein said factor required for sustainedself-renewal is selected from the group consisting of transforminggrowth factor α, epidermal growth factor, and hepatocyte growth factor.